Poxviruses are complex DNA viruses that encode up to 200 genes. Variola virus (VaV) was the causative agent of smallpox, one of the most devastating human diseases that was eradicated as a result of the use of vaccinia virus (VV) as a smallpox vaccine in the WHO global eradication campaign. Cowpox virus (CPV) is related to VV and is thought to be a rodent virus that causes sporadic infections in a number of mammals. Ectromelia virus (EV) is a natural mouse pathogen and the causative agent of mousepox, a generalized mouse disease with similarities to human smallpox.
The immune response has evolved as an efficient mechanism of protection from infection by pathogens such as viruses. To replicate in the immunocompetent host, viral mechanisms to evade the immune response have evolved (Alcami & Koszinowski, 2000). Poxviruses encode a broad variety of proteins that counteract the host immune response (Alcami, 2003, Seet et al., 2003). One of the immunomodulatory mechanisms encoded by poxviruses is the production of secreted proteins that bind cytokines, a family of proteins that regulate the immune response.
Four secreted TNFRs encoded by poxviruses have been described, and named Cytokine response modifier B (CrmB), CrmC, CrmD and CrmE (Hu et al., 1994, Loparev et al., 1998, Saraiva & Alcami, 2001, Smith et al., 1996). These proteins have amino acid sequence similarity to the cysteine-rich domains (CRDs) present in the human TNFRs and constituting the TNF binding extracellular domain. The viral proteins lack the transmembrane and intracellular domains of the cellular TNFRs (FIGS. 1 and 2).
All four vTNFRs have been shown to be secreted from virus-infected cells, to bind TNF and to block TNF biological activity. The CRDs of vTNFRs are predicted to bind TNF, and this has been demonstrated for the CrmB homologue encoded by myxoma virus (M-T2) (Schreiber et al., 1997), and our own experiments showing that the three N-terminal CRDs of CrmD encode TNF binding and inhibitory activity (see below).
Representative members of two of the vTNFRs named CrmB and CrmD are: CPV CrmB (SEQ ID NO 11 and 12), VaV CrmB (SEQ ID NO 9 and 10), CPV CrmD (SEQ ID No 1 and 2) and EV CrmD (SEQ ID NO 3 and 4). CrmB and CrmD have an additional CTD with no amino acid sequence similarity to cellular proteins in the databases (FIGS. 1 and 2). This domain is not required for TNF binding and its function has not been defined. Three open reading frames (ORFs) encoded by poxviruses have amino acid sequence similarity to the CTD of vTNFRs. Representative members of these ORFs are EV E12 (CTD1) (SEQ ID NO 5 and 6), EV E184 (CTD2) (SEQ ID NO 19 and 20), and CPV B21R/V218 (CTD3) (Accession No. O72758 and SEQ ID NO 13 and 14) (FIGS. 1 and 2).
A number of secreted proteins that bind chemokines have been described in several poxviruses and herpesviruses (Alcami et al., 1998, Bryant et al., 2003, Graham et al., 1997, Lalani et al., 1997, Parry et al., 2000, van Berkel et al., 2000) (Table 1). These virus-encoded chemokine binding proteins (vCKBPs) have no amino acid sequence similarity to the cellular seven-transmembrane-domain chemokine receptors or other cellular proteins (Alcami, 2003, Seet et al., 2003, Seet & McFadden, 2002). Structural and functional studies on the 35 kDa vCKBP encoded by CPV and M3 encoded by murine gammaherpesvirus 68 (MHV-68) have demonstrated that these viral proteins represent novel protein domains or structures that have the ability to bind chemokines (Alexander et al., 2002, Carfi et al., 1999). The ability of some of the vCKBPs to block leukocyte migration into infected tissues and viral pathogenesis has been demonstrated (Bridgeman et al., 2001, Graham et al., 1997, Johnston & McFadden, 2004, Lalani et al., 1999, Reading et al., 2003).
The C-terminal domain (CTD) of the viral TNF receptors (vTNFRs) cytokine response modifier B (CrmB) and CrmD have no ascribed function to date and no sequence similarity to host proteins. We found that this domain confers these vTNFRs the ability to bind several chemokines, and that the CTD expressed independently of the TNF binding domain of vTNFRs binds chemokines. This protein domain is also found in three additional poxvirus-encoded proteins predicted to be secreted, and we show that two of them encoded by the E12 gene of ectromelia virus (EV) and B21R gene of cowpox virus (CPV) bind chemokines. We propose that the CTD of vTNFRs defines a novel structural domain that binds chemokines. These proteins may modulate chemokine activity in vivo and be used to modulate adverse immune and inflammatory responses in a number of human disease conditions. The expression of this CTD fused to soluble TNFRs may enhance the immunomodulatory properties of TNFRs already used in the clinic. In addition, the CTD enhance the TNF binding activity of the N-terminal cysteine-rich domains of vTNFRs, and vTNFRs bind other members of the TNF ligand superfamily.